CUT&RUN - Improved Method to Study Protein-DNA Interactions
CUT&RUN, which stands for cleavage under targets and release using nuclease offers a new approach to pursue epigenetics1.
CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling1.
However it is not limited to profiling; CUT&RUN has the potential to replace all ChIP-based applications.
Have a look at our video to get an introduction to the topic CUT&RUN!
CUT&RUN Advantages - Combining Perks of ChIP-Seq Variations
As CUT&RUN is performed on intact cells or nuclei without fragmentation, it can be used to probe all genomic compartments. The cleaved chromatin complexes diffuse out of the nuclei where they can be harvested in supernatant. The rest of the undigested genome is retained in the intact nuclei. For ChIP-Seq on the other hand, the majority of chromatin is sheared or digested resulting in comparison in a worse signal-to-noise ratio. Consequently, ChIP-Seq requires more sequencing depth and with each sequencing run additional sample amount, labour time and money.
CUT&RUN Workflow: Extraction without fragmentation
The CUT&RUN is straightforward and can be completed in under a day using standard lab equipment. CUT&RUN is suitable for application down to 100 cells for profiling H3K27me3 or 1000 cells for CTCF sequence-specific DNA- binding protein1.
Therefore, CUT&RUN enables targeted genome-wide maps of protein- DNA interactions even for rare cell types. In comparison with XChiP-seq data both CUT&RUN runs reveal reliable peaks with less background noise. One of the strengths of CUT&RUN is that the entire reaction is performed in situ.
CUT&RUN: Frequently Asked Questions
- What are the advantages and disadvantages of CUT&RUN and CUT&Tag compared to ChIP-seq? How do I choose between both methods.
- Why is the DNA yield so low?
- How do I choose a primary antibody for CUT&RUN or CUT&Tag?
- Why do I need a negative control antibody for CUT&RUN? Why not just use a no-antibody control?
- Can I replace the antibody negative control for CUT&RUN using a knock-out (or knock-down) of my protein?
- Do I need to use a secondary antibody for CUT&RUN and CUT&Tag?
- One of my antibodies is mouse. Has your pAG-MNase good affinity for mouse antibodies, or do you advise to use a rabbit anti-mouse secondary antibody?
- Should I include heterologous spike-in DNA for quantitation?
- Is it possible to fix the cells prior to immobilization?
- Is it possible to use the antibodies-online CUT&RUN product sets with plant tissue samples?
- Is it possible to adapt CUT&RUN to RNA binding proteins?
- Instead of the proteinase K digestion can I denature the proteins in the CUT&RUN product complexes by heat?
- What is preferable for DNA extractions prior to library preparation: extraction using phenol-chloroform or affinity purification using a column?
- Is it possible to do single-end instead of paired-end sequencing of the CUT&RUN libraries?
What are the advantages and disadvantages of CUT&RUN and CUT&Tag compared to ChIP-seq? How do I choose between both methods.
Advantages of CUT&RUN and CUT&Tag compared to ChIP-seq are a better signal-to-noise ratio, higher sensitivity, a wider dynamic range, a lower requirement of sequencing reads and cell number.
CUT&Tag has the advantage that the sequencing primers are being attached to the cleaved DNA fragments and requires fewer library preparation steps than CUT&RUN. No additional annealing is necessary. The method works particularly well for nucleosomal and tightly bound proteins. It has also been streamlined by the Henikoff lab into a protocol where the entire process takes place in one tube and high throughput variations amenable for automation are available.
CUT&RUN on the other hand is preferable for transcription factors and other less tightly bound DNA binding proteins that are sensitive to the higher salt concentration in CUT&Tag necessary to prevent off-target tagmentation of accessible chromatin by Tn5. In addition, the spatial resolution of the MNase digestion is higher than that of the tagmentation, enabling a clearer footprint of the protein of interest.
Why is the DNA yield so low?
CUT&RUN and CUT&Tag are performed using low cell numbers and the background signal is considerably lower than e.g. for ChIP. This can make reliable measurements of the DNA concentration using a fluorometric assay or by capillary electrophoresis challenging.
To assess the success of the CUT&RUN and CUT&Tag methods it is recommended to include a reaction using an antibody against and abundant histone modification such as h4K27me ( ABIN6923144) or h4K4me3 ( ABIN2668472) as a positive control. DNA fragments prepared using such an antibody can be measured by capillary electrophoresis on a Bioanalyzer or Tapestation or fluorometrically on a Qubit or Nanodrop fluorometer.
How do I choose a primary antibody for CUT&RUN or CUT&Tag?
Antibodies that are recommended for ChIP-seq do not necessarily work in CUT&RUN in CUT&Tag. In contrast to ChIP-seq, the antigen is generally in its native state without additional fixation. Unless an antibody has already been tested for CUT&RUN/Tag, a recommendation for a method in which the antigen is expected to be in a native state is helpful, e.g. Immunofluorescence. Unless indicated otherwise, the recommended dilution for immunofluorescence is also a good starting point for the antibody’s concentration in CUT&RUN/Tag.
Why do I need a negative control antibody for CUT&RUN? Why not just use a no-antibody control?
The MNase used for CUT&RUN is an endo- and exonuclease that will unspecifically bind and cleave unprotected DNA in hyper-accessible DNA, e.g. in regions surrounding regulatory elements. Free MNase will preferentially cut DNA within these hyper-accessible regions, thus potentially causing false positives and increase the background signal in general.
To avoid this undesired effect of untethered MNase, chromatin is randomly coated with the CUT&RUN guinea pig anti-rabbit IgG negative control antibody ( ABIN6923140) prior to the addition of pAG-MNase ( ABIN6950951) to the samples. pAG-MNase is then tethered via its Protein A or Protein G portion to the antibody’s Fc fragment and background DNA fragmentation is dictated by the random antibody binding as opposed to the nuclease digestion of hyper-accessible DNA regions.
Can I replace the antibody negative control for CUT&RUN using a knock-out (or knock-down) of my protein?
Both controls are useful but address different aspects of the experiment and are therefore not interchangeable.
The CUT&RUN guinea pig anti-rabbit IgG negative control antibody ( ABIN6923140) is used to establish a reference background for peak calling. This is necessary because of the sparse background signal in CUT&RUN samples compared to ChIP-seq samples. The ko (or kd) control on the other hand gives an impression of unspecific binding of the antibody directed against the protein of interest to other proteins. It is useful to avoid identification of false positive signals.
Do I need to use a secondary antibody for CUT&RUN and CUT&Tag?
Depending on the host species and isotype of the antibody and the Protein A and/or Protein G MNase fusion protein, a secondary antibody may be necessary for MNase binding. Protein A has good high affinity to all rabbit IgG antibodies but low affinity to rat, goat and sheep IgG isotype antibodies and certain mouse IgG antibody subclasses, in particular IgG1. Protein G on the other hand binds well to the Fc region of mouse, goat, sheep, and most rat IgG. Its affinity to rabbit IgG however is lower than that of Protein A. When using pAG-MNase introduced with the improved CUT&RUN protocol it is therefore generally not necessary to use a secondary antibody. Use of the pA-MNase of the original protocol however might require the use of a secondary antibody raised in rabbit to assure efficient binding of the fusion protein to the antibody.
For CUT&Tag a secondary antibody is recommended to increase the local concentration of Fc fragment binding site in the vicinity of the intended transposition site around the antigen of interest. This step is necessary to increase the specific signal.
One of my antibodies is mouse. Has your pAG-MNase good affinity for mouse antibodies, or do you advise to use a rabbit anti-mouse secondary antibody?
The pAG-MNase will work well with your murine antibody. The addition of protein G to the MNase is primarily to accomodate the use of mouse IgG1 monoclonal antibodies that bind poorly to protein A. The other IgG isotypes bind well to either protein A or protein G.
Should I include heterologous spike-in DNA for quantitation?
Our protocol is largely based on the improved CUT&RUN protocol. Here, the authors show that accurate quantitation is possible using heterologous spike-in DNA or carry-over E. coli DNA from the pAG-MNase purification. Therefore, the addition of heterologous spike-in DNA is not necessary.
Is it possible to fix the cells prior to immobilization?
It is possible to fix your samples, e.g. to avoid dissociation of larger protein complex from the DNA during the course for the experiment. You can either follow your established cross-linking procedure or mild cross-linking conditions using formaldehyde at a lower concentration of 0.1%. Cross-linking at 1% formaldehyde can actually reduce signal, possibly due to epitope masking. In these cases, a lower concentration of cross-linker is preferable.
Is it possible to use the antibodies-online CUT&RUN product sets with plant tissue samples?
The CUT&RUN method can be applied to plant tissue samples. An essential step in addition to those lined out in the protocol is the generation of spheroblasts so that it becomes possible to permeabilize the plasma membrane for the application of the antibodies and the MNase fusion protein. Alternatively, use isolated nuclei as sample material.
The CUT&RUN rabbit anti-h4K27me3 positive control antibody ( ABIN6923144) and the CUT&RUN guinea pig anti-rabbit IgG negative control antibody ( ABIN6923140) as well as the ConA beads ( ABIN6923139 or ABIN6952467) are suitable for use with plant samples.
Is it possible to adapt CUT&RUN to RNA binding proteins?
The MNase used for CUT&RUN also accepts RNA as substrate so it might be possible to adapt the CUT&RUN protocol for use on RNA binding proteins.
RNA in the cytoplasm will attract the degradation machinery if it is lacking the 5' cap and the 3' poly-A tail. Therefore, I would suggest to use work with isolated nuclei. This has the additional benefit, that you can omit the digitonin in the buffers, which is used to replace the cholesterol and permeabilize the cell membrane. The isolated nuclei may then be immobilized using magnetic ConA beads like for a CUT&RUN experiment. An antibody against the protein of interest is added and subsequently the pAG-MNase is tethered to the antibody, thus bringing the MNase into proximity of the RNA of interest. Isolated RNA can then be reverse transcribed into cDNA to produce your sequencing library.
In order to dispose of any contaminating DNA include a DNase treatment in the protocol subsequently to the RNA-prep and before the library prep. Normalization based on the E. coli DNA carried over with the MNase or a spike-in DNA is not an option in this case. Instead, consider using the total read numbers to normalize across samples or include a reference RNA of a known concentration.
Instead of the proteinase K digestion can I denature the proteins in the CUT&RUN product complexes by heat?
We recommend against this option: the DNA of interest is at his point present in a complex consisting of the DNA, the antigen, the corresponding antibody, and the pAG-MNase. Boiling this complex will likely precipitate the DNA together with denatured protein. This will also primarily affect the short CUT&RUN products and not the larger DNA molecules, leading to a decreased signal to noise ratio in your library and potentially also reducing the library’s complexity. This effect is further exacerbated because of the lower melting temperature of these short molecules compared to the longer contaminating DNA molecules.
What is preferable for DNA extractions prior to library preparation: extraction using phenol-chloroform or affinity purification using a column?
A potential issue when using SPRI beads for the DNA fragment clean-up is the carry-over of active Proteinase K, which can interfere with the downstream PCR amplification. Therefore, a phenol-chloroform extraction is preferable to assure complete denaturation of Proteinase K.
Is it possible to do single-end instead of paired-end sequencing of the CUT&RUN libraries?
Single-end sequencing instead of paired-end sequencing is possible. However, it has drawbacks compared to paired-end sequencing: (i) For abundant targets like histone marks or transcription factors a large number of binding sites is expected. Paired-end sequencing facilitates unambiguous mapping to the correct genomic position. This additional information reduces the necessary sequencing depth. (ii) MNase will digest the target DNA until the section covered by the protein of interest. Paired-end sequencing will reveal this footprint while the information is lost in single-end sequencing.
Tips for Antibody Selection
The successful execution of cleavage under target and release using nuclease (CUT&RUN) hinges on antibody selection. A factor- or histone-specific antibody is bound to chromatin in situ followed by binding to the antibody of a protein A-micrococcal nuclease (pA-MNase) fusion. As is the case with ChIP, the success depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. We compiled a list of primary and secondary antibodies suitable for CUT&RUN down below. Addtionally, antibodies-online supports the validation of antibodies for CUT&RUN. You can participate in our independent validation initiative (IVI). Propose and perform a validation experiment and get a full refund on the validated antibody. Please contact us, if you are interested in validating one of our CUT&RUN products!
Which secondary antibodies can I use for CUT&RUN?
Which primary antibodies can I use for CUT&RUN?
- Skene PJ and Henikoff S. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature (2018) PMID 25652980
- Brahma S and Henikoff S. RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast. Mol. Cell (2018) PMID 30554944
- Hainer SJ et al. Profiling of Pluripotency Factors in Single Cells and Early Embryos. Cell (2019) PMID 30955888
- Kaya-Okur H et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature Communications (2019) PMID 31036827
- Meers MP et al. Improved CUT&RUN chromatin profiling tools. eLife (2019) PMID 31232687
- Kaya-Okur H et al. Efficient low-cost chromatin profiling with CUT&Tag. Nature Protocols (2020) PMID 32913232
- Henikoff S. et al. Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. eLife (2020) PMID 33191916
Cut and Run, CutAndRun, CUT and Tag, CutAndTag, ChIP-seq, ChIP-sequencing, chromatin; chromosomes; epigenomics; gene expression; genetics; genomics; human; spike-in calibration; in situ profiling; transcription factors; A-Micrococcal Nuclease